IL-7R/CD127 (Phospho-Tyr449) Colorimetric Cell-Based ELISA Kit
- SKU:
- CBCAB01457
- Product Type:
- ELISA Kit
- ELISA Type:
- Cell Based Phospho Specific
- Reactivity:
- Human
- Reactivity:
- Mouse
- Detection Method:
- Colorimetric
Description
| Product Name: | IL-7R/CD127 (Phospho-Tyr449) Colorimetric Cell-Based ELISA |
| Product Code: | CBCAB01457 |
| ELISA Type: | Cell-Based |
| Target: | IL-7R/CD127 (Phospho-Tyr449) |
| Reactivity: | Human, Mouse |
| Dynamic Range: | > 5000 Cells |
| Detection Method: | Colorimetric 450 nm |
| Format: | 2 x 96-Well Microplates |
The IL-7R/CD127 (Phospho-Tyr449) Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect IL-7R/CD127 protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated IL-7R/CD127 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on IL-7R/CD127 phosphorylation.
Qualitative determination of IL-7R/CD127 (Phospho-Tyr449) concentration is achieved by an indirect ELISA format. In essence, IL-7R/CD127 (Phospho-Tyr449) is captured by IL-7R/CD127 (Phospho-Tyr449)-specific primary (1ø) antibodies while the HRP-conjugated secondary (2ø) antibodies bind the Fc region of the 1ø antibody. Through this binding, the HRP enzyme conjugated to the 2ø antibody can catalyze a colorimetric reaction upon substrate addition. Due to the qualitative nature of the Cell-Based ELISA, multiple normalization methods are needed:
| 1. | A monoclonal antibody specific for human GAPDH is included to serve as an internal positive control in normalizing the target absorbance values. |
| 2. | Following the colorimetric measurement of HRP activity via substrate addition, the Crystal Violet whole-cell staining method may be used to determine cell density. After staining, the results can be analysed by normalizing the absorbance values to cell amounts, by which the plating difference can be adjusted. |
| Database Information: | Gene ID: 3575, UniProt ID: P16871, OMIM: 126200/146661/608971, Unigene: Hs.591742/Hs.635723 |
| Gene Symbol: | IL7RA |
| Sub Type: | Phospho |
| UniProt Protein Function: | IL7R: Receptor for interleukin-7. Also acts as a receptor for thymic stromal lymphopoietin (TSLP). Defects in IL7R are a cause of severe combined immunodeficiency autosomal recessive T-cell-negative/B-cell- positive/NK-cell-positive (T(-)B(+)NK(+) SCID). A form of severe combined immunodeficiency (SCID), a genetically and clinically heterogeneous group of rare congenital disorders characterized by impairment of both humoral and cell-mediated immunity, leukopenia, and low or absent antibody levels. Patients present in infancy recurrent, persistent infections by opportunistic organisms. The common characteristic of all types of SCID is absence of T-cell-mediated cellular immunity due to a defect in T-cell development. Genetic variations in IL7R are a cause of susceptibility to multiple sclerosis type 3 (MS3). A multifactorial, inflammatory, demyelinating disease of the central nervous system. Sclerotic lesions are characterized by perivascular infiltration of monocytes and lymphocytes and appear as indurated areas in pathologic specimens (sclerosis in plaques). The pathological mechanism is regarded as an autoimmune attack of the myelin sheat, mediated by both cellular and humoral immunity. Clinical manifestations include visual loss, extra-ocular movement disorders, paresthesias, loss of sensation, weakness, dysarthria, spasticity, ataxia and bladder dysfunction. Genetic and environmental factors influence susceptibility to the disease. A polymorphism at position 244 strongly influences susceptibility to multiple sclerosis. Overtransmission of the major 'C' allele coding for Thr-244 is detected in offspring affected with multiple sclerosis. In vitro analysis of transcripts from minigenes containing either 'C' allele (Thr-244) or 'T' allele (Ile-244) shows that the 'C' allele results in an approximately two-fold increase in the skipping of exon 6, leading to increased production of a soluble form of IL7R. Thus, the multiple sclerosis associated 'C' risk allele of IL7R would probably decrease membrane-bound expression of IL7R. As this risk allele is common in the general population, some additional triggers are probably required for the development and progression of MS. Belongs to the type I cytokine receptor family. Type 4 subfamily. 4 isoforms of the human protein are produced by alternative splicing. |
| UniProt Protein Details: | Protein type:Membrane protein, integral; Receptor, cytokine Chromosomal Location of Human Ortholog: 5p13 Cellular Component: external side of plasma membrane; extracellular region; integral to membrane; plasma membrane Molecular Function:antigen binding; interleukin-7 receptor activity; protein binding Biological Process: B cell proliferation; cell growth; cell morphogenesis; cell surface receptor linked signal transduction; homeostasis of number of cells; immune response; immunoglobulin production; lymph node development; negative regulation of T cell mediated cytotoxicity; positive regulation of T cell differentiation in the thymus; regulation of cell size; regulation of DNA recombination; signal transduction; T cell differentiation Disease: Severe Combined Immunodeficiency, Autosomal Recessive, T Cell-negative, B Cell-positive, Nk Cell-positive |
| NCBI Summary: | The protein encoded by this gene is a receptor for interleukin 7 (IL7). The function of this receptor requires the interleukin 2 receptor, gamma chain (IL2RG), which is a common gamma chain shared by the receptors of various cytokines, including interleukins 2, 4, 7, 9, and 15. This protein has been shown to play a critical role in V(D)J recombination during lymphocyte development. Defects in this gene may be associated with severe combined immunodeficiency (SCID). Alternatively spliced transcript variants have been found. [provided by RefSeq, Dec 2015] |
| UniProt Code: | P16871 |
| NCBI GenInfo Identifier: | 215274000 |
| NCBI Gene ID: | 3575 |
| NCBI Accession: | P16871.2 |
| UniProt Secondary Accession: | P16871,Q05CU8, Q6NSP4, Q6NWM0, Q6NWM1, Q6NWM2, Q6NWM3 Q6SV45, Q9UPC1, B2RCS6, B4DVT1, |
| UniProt Related Accession: | P16871 |
| Molecular Weight: | 28,724 Da |
| NCBI Full Name: | Interleukin-7 receptor subunit alpha |
| NCBI Synonym Full Names: | interleukin 7 receptor |
| NCBI Official Symbol: | IL7R |
| NCBI Official Synonym Symbols: | ILRA; CD127; IL7RA; CDW127; IL-7R-alpha |
| NCBI Protein Information: | interleukin-7 receptor subunit alpha |
| UniProt Protein Name: | Interleukin-7 receptor subunit alpha |
| UniProt Synonym Protein Names: | CDw127; CD_antigen: CD127 |
| Protein Family: | Interleukin-7 receptor |
| UniProt Gene Name: | IL7R |
| UniProt Entry Name: | IL7RA_HUMAN |
| Component | Quantity |
| 96-Well Cell Culture Clear-Bottom Microplate | 2 plates |
| 10X TBS | 24 mL |
| Quenching Buffer | 24 mL |
| Blocking Buffer | 50 mL |
| 15X Wash Buffer | 50 mL |
| Primary Antibody Diluent | 12 mL |
| 100x Anti-Phospho Target Antibody | 60 µL |
| 100x Anti-Target Antibody | 60 µL |
| Anti-GAPDH Antibody | 60 µL |
| HRP-Conjugated Anti-Rabbit IgG Antibody | 12 mL |
| HRP-Conjugated Anti-Mouse IgG Antibody | 12 mL |
| SDS Solution | 12 mL |
| Stop Solution | 24 mL |
| Ready-to-Use Substrate | 12 mL |
| Crystal Violet Solution | 12 mL |
| Adhesive Plate Seals | 2 seals |
The following materials and/or equipment are NOT provided in this kit but are necessary to successfully conduct the experiment:
- Microplate reader able to measure absorbance at 450 nm and/or 595 nm for Crystal Violet Cell Staining (Optional)
- Micropipettes with capability of measuring volumes ranging from 1 µL to 1 ml
- 37% formaldehyde (Sigma Cat# F-8775) or formaldehyde from other sources
- Squirt bottle, manifold dispenser, multichannel pipette reservoir or automated microplate washer
- Graph paper or computer software capable of generating or displaying logarithmic functions
- Absorbent papers or vacuum aspirator
- Test tubes or microfuge tubes capable of storing ≥1 ml
- Poly-L-Lysine (Sigma Cat# P4832 for suspension cells)
- Orbital shaker (optional)
- Deionized or sterile water
*Note: Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.
| Step | Procedure |
| 1. | Seed 200 µL of 20,000 adherent cells in culture medium in each well of a 96-well plate. The plates included in the kit are sterile and treated for cell culture. For suspension cells and loosely attached cells, coat the plates with 100 µL of 10 µg/ml Poly-L-Lysine (not included) to each well of a 96-well plate for 30 minutes at 37 °C prior to adding cells. |
| 2. | Incubate the cells for overnight at 37 °C, 5% CO2. |
| 3. | Treat the cells as desired. |
| 4. | Remove the cell culture medium and rinse with 200 µL of 1x TBS, twice. |
| 5. | Fix the cells by incubating with 100 µL of Fixing Solution for 20 minutes at room temperature. The 4% formaldehyde is used for adherent cells and 8% formaldehyde is used for suspension cells and loosely attached cells. |
| 6. | Remove the Fixing Solution and wash the plate 3 times with 200 µL 1x Wash Buffer for five minutes each time with gentle shaking on the orbital shaker. The plate can be stored at 4 °C for a week. |
| 7. | Add 100 µL of Quenching Buffer and incubate for 20 minutes at room temperature. |
| 8. | Wash the plate 3 times with 1x Wash Buffer for 5 minutes each time. |
| 9. | Add 200 µL of Blocking Buffer and incubate for 1 hour at room temperature. |
| 10. | Wash 3 times with 200 µL of 1x Wash Buffer for 5 minutes each time. |
| 11. | Add 50 µL of 1x primary antibodies Anti-IL-7R/CD127 (Phospho-Tyr449) Antibody, Anti-IL-7R/CD127 Antibody and/or Anti-GAPDH Antibody) to the corresponding wells, cover with Parafilm and incubate for 16 hours (overnight) at 4 °C. If the target expression is known to be high, incubate for 2 hours at room temperature. |
| 12. | Wash 3 times with 200 µL of 1x Wash Buffer for 5 minutes each time. |
| 13. | Add 50 µL of 1x secondary antibodies (HRP-Conjugated AntiRabbit IgG Antibody or HRP-Conjugated Anti-Mouse IgG Antibody) to corresponding wells and incubate for 1.5 hours at room temperature. |
| 14. | Wash 3 times with 200 µL of 1x Wash Buffer for 5 minutes each time. |
| 15. | Add 50 µL of Ready-to-Use Substrate to each well and incubate for 30 minutes at room temperature in the dark. |
| 16. | Add 50 µL of Stop Solution to each well and read OD at 450 nm immediately using the microplate reader. |
(Additional Crystal Violet staining may be performed if desired – details of this may be found in the kit technical manual.)
